Antipressor and method of making



Patented Jan. 15, 1946 ANTIPRESSOR AND METHOD OF MAKING Harold M. Rabinowltz, Brooklyn, N. Y.

Serial No. 477,161

No Drawing. Application February 25, 1943,

9 Claims.

This invention relates to an antipressor and the method of making it.

I The antipressor is particularly useful in lowering the blood pressure of human beings in cases comprising the ethyl acetate soluble extract of urine of a liver-bearing animal, the extract containing benzoic acid and the urobilinogen type of chromogen normally present in the urine and ing preferably substantially free of fat.

Urobilinogen is ordinarily considered to be of the formula C3sH44O6N4, the nitrogen being largely at least in the form of substituted amine groups =NH. Chromogens of the urobilinog'en type are a. well recognized class of compounds of composition and properties closely related to urobilinogen.

It will be understood that. the benzoic acid and urobilinogen type chromogens in my composition are combined to a substantial extent, presumably as a salt of the benzoic acid with substituted amine grouping in the urobilinogen. It isv not necessary to the invention that they be completely combined, it being necessary only that the two be mixed together before use so that the reaction between them may occur to the extent that is characteristic of the two materials when mixed.

The proportions of the. two materials is not critical. However, the antipressor composition should contain not less than the amounts of the Y benzoic acid and urobilinogen, either free or.

combined, that give positive reactions in Ehrlich's test for urobilinogen (Hawk and Bergeim,

Practical Physiological Chemistry, 11th. ed., p. 757) and a positive reaction in the Fleury test for benzoic acid, as

Suitable proportions that may be used are approximately 0.5 to 2.5 parts of the urobilinosen for 100 parts of benzoic acid, particularly satisfactory results being obtained when the proportion the urobilinozen about 1 to 2 parts. when the proportion of urobilinosen is substantially less than 0.5 part, then the dosage or the described in Merck's Index under whole composition must be increased. When.

the proportion of the urobilinogen is much in excess oi. 2.5 parts, then there is introduced unnecessary expense in the cost per unit of weight of the antipressor composition.

The antipressor composition of the kind de-- scribed and consisting of benzoic acid and a chromogen of the urobilinogen type, that is a fat-soluble urobilinogen, will be sometimes hereinafter referred to, for convenience, as Benzochrome.

Numerous cases of essential hypertension of all grades have :been treated with Benzochrome. In a typical treatment the Benzochrome is administered in doses of to 150 mg. dissolved or dispersed thoroughly in a dispersion medium. Thus there may be used a dispersion of the Benzochrome in warm almond oil in the proportion of about 25 to 50 mg. of the former to 1 cc. of the oil. The almond oil solution is injected intramuscularly. In an alternative procedure the Benzochrome is injected intravenously. In this case the Benzochrome in the same dosage amounts as above is first dissolved in water or a physiologic saline solution as, for example, by

dissolving 0.1 part of the Benzochrome in state. The results were satisfactory. with comparts of distilled water or oi physiologic salt solution. The Benzochrome not only lowers the blood pressure levels but also relieves the symptoms accompanying essential hypertension and eclampsia.

The maximum efiect with regard to the lowering of both systolic and diastolic levels is obtained within about 48 to 72 hours after injection. en administered in such doses of about 75 to v mg. of the'activeantipressor in almond oil plete termination of the eclamptic state.

Tests have shown that neither benzoic acid nor urobilinogen alone are satisfactory in lowering blood pressure levels. when there is used, however, the mixed benzoic acid and a chromogen of the urobilinozen type in doses ranging from. say, 50 to 200 man. there is ordinarily a. very marked fall in blood pressure within 48 hours with a concomitant alleviation of secondary symptoms.

The active antipressor or Benzochrome is diluted before use, as with the almond oil or water, as described above. In place of the almond oil as diluting medium when the Benzochrome is to be introduced intramuscularly there may be used other oils as, for example, sesame, olive, or peanut oil. .The oil solutions are particularly satisfactory because they show when introduced into the system only a very slow rate of absorption of the Benzochrome. The effect is not an initial strong surge but a prolonged desirably steady eiiect. In place of the, oil described as the dispersion medium, there may be used lecithin in fluid form. The dispersionliquid that is used should have appreciable solvent power for Benzochrome and be non-irritating and non-injuri- -ous when introduced intramuscularly or intravenousiy.

In making the antipressor of this invention, the benzoic acid and a urobilinogen may be prepared separately in any convenient manner and then compounded in proportions to show both the Ehrlich test for. urobilinogen and the Fleury test for benzoic acid or in the typical proportions stated numerically above.

I have found, however an economical source of a mixture of the two ingredients that are essential to my antipressor composition. This source is urine of liver-bearing animals as, for

example, horses, cows, or sheep. Th urine from horses and particularly from geldings is preferred because of its relative richness in the necessary ingredients for my antipressor composition as well as its availability.

In making the antipressor composition from urine, the urine is first treated to remove ordinary fats, as by solvent extraction in the presence of a large proportion of water such as the normal water content of urine; These fats so removed are then discarded. The remainder of the urine is subjected to treatment to provide a fatsoluble extract, as, for example, to evaporation followed by extraction with ethyl acetate or the like. The extracted material may be freed of the extracting solvent and then is preferably subdected to further purification, including crystallization from a warm solution in a volatile solvent i'or the benzoic acid and urobilinogen. The resulting antipressor is substantially free of fat.

In removing the ordinary andundesired fats from urine before extracting the desired benzoic acid and chromogen of urobilinogen type, there is used any suitable extracting medium for fats,

as for example, butyl ether, amyl ether, hexyl acetate or like solvent for fats that is practically insoluble in water and is easily separated therefrom by difierenoe of specific gravity.

Preferably the urine is first made slightly acid, as by addition oil's. small amount of acetic,

, propionic, or hydrochloric acid in proportion to establish the pH at aboutthe neutral point or on the acid side. While various pH's may be used,

.a pH of about 6 to 7 is satisfactory. The acidified urine is then contacted thoroughly with the is thenextraoted with ethyl acetate, ethyl ether,

solvent. The residue so obtained is a crude form of the desired antipressor consisting principally of benzoic acid and a chromogen of the urobilinogen type including their reaction product. It may be further purified by dissolving to as large a concentration as feasible in distilled water at about C., filtering the resulting solution and cooling the filtrate to cause crystals of the antipressor material to form. These crystals may be separated, dried, and ground. They are then ready to be dissolved in sweet almond oil, a saline solution, or other medium preparatory to injection. These crystals include a compound of urobilinogen and benzoicacid.

The product (Benzochrome) gives a positive color reaction for urobilinogen with Ehrlichs' test and a positive reaction for benzoic acid by the Fleury test. It may contain an appreciable amount of urobilin in desired proportion, say in proportion about equal to the urobilinogen. The product possesses .the property of fluorescence. Under the influence of ultra violet rays, it phosphoresces. It is soluble in ethyl ether, chloroform, ethyl acetate, and similar organic solvents but only slightly soluble in cold water. It melts at about 121 to 123 0., the exact temperature within the approximate range stated depending upon the purity. Less pure forms melt at about 118.6 to 121 C. y

The method of manufacture will be further illustrated by detailed description in connection with the following specific examples.

Example I To three gallons of horse urine acidulated with acetic acid, two gallons of butyl ether are added, the mixture agitated for four hours at 5000 R. P. M, and then allowed to separate into two layers. The upper brown gelatinous layer (fat extract) is discarded and the lower dark layer of fluid is used in preparing Benzochrome. This lower layer is heated at a temperature of 175 F., slowly evaporated to a black mass, and then dried. The dried dark solid mass is extracted with ethyl acetate. The extract is filtered and the filtrate evaporated todryness. The dried material is extracted with a'mixture of equal partsof chloroform and alcohol and illtered. The filtered extract so obtained is thoroughly agitated with suificient distilled water to separate the alcohol from the mixture. The chloroform layer is removed, evaporated to. dryness, and the residue dried. The dried material is a dark tobacco-colored substance.

This residue is extracted with distilled water at a temperature of 90 C. The hot solution is poured through filter paper. The filtrate iscollected and allowed 'to cool for 24 hours. An ecru-colored material, precipitated from the filtrate, is collected on filter paper and thoroughly dried. This material thus precipitated and colflected on the filter contains the combination of the benzoic acid and the chromogen of the urobilinogen type and constitutes the chief fraction oi antipressor material obtained from the urine.

; Example II p The remaining clear faintly yellow fluid (mother liquor) separated from the said ecru-colored material in Example I, is evaporated under negative pressure. This produces a rust-colored dry substance. which gives a marked positive color reaction for urobilinogen with Ehrlichs reagent I and contains a very concentrated form of the urobilinogen type of chromogen.

Equal parts byweight of this concentrated urobilinogen type of chromogenand benzoic acid are dissolved in distilled water and heated to a temperature of 90 C., the whole being thoroughly mixed while heating. The hot solution is poured through filter paper and the filtrate allowed to cool and stand for 24 hours. An ecrucolored material, precipltated from the filtrate, is collected on a filter paper and thoroughly dried.

I This dried substance is composed of benzoic acid and a chromogen ofthe urobilinogen type,

i free or combined. It gives a positive color reaction for urobilinogen with Ehrlich's reagent and a positive reaction for benzoic acid with the Fleury test. It reveals no absorption spectra and possesses the properties of fluorescence to a ca-v nary yellow color and phosphorescence with ultraviolet ray. It is soluble in ether and chloroform and slightly solublein cold water. It shows a definite antipressor property when dissolved in warm sweet almond oil and administered intramuscularly and aflords definite relief of symptoms in essential hypertension. The physical,

chemical and therapeutic properties are identical with that of the previously described benzoic acid and chromogen compound referred to as Benzochrome.

The same process is repeated for the faintly yellow colored filtrate with consequent recovery of. additional urobilinogen.

This application is a continuation in part of my copendingapplication Ser. No.'437,843 filed April 6, 1942, for Therapeutic agent for the treatment of hypertension.

for urobilinogen and a positive Fleury test ior benzoic acid whenthe tests'are applied to the antipressor; 4 I

3. The method of making an antipressor which comprises extracting fat from the urine of a liver bearing animal by means of a fat solvent that is a non-solvent for the urobilinogen present in such urine, evaporating the remainder of the urine to a substantially dry residue, extracting the residue with a solventfor urobilinogen, separating the resulting extract from the undissolved material, evaporating the said solvent irom the extract, and adding benzoic acid, the product so formed being the antipressor composition.

4. The method described inclaim 8, including recrystallizing from hot water the said antipressor composition.

5. An antipressor composition comprising the products of the reaction of benzoic acid with the urobilinogen fraction of the urine of a liver hearing animal, the said fraction being the substantiallyfat free, ethyl acetate soluble material oi the said urine.

6. The method oi making an antipressor composition which comprises acidifying the urine of, a liver bearing animal, extracting fat irom the urine by means or a fat solvent that is a nonsolvent for the urobilinogen present in such urine, evaporating the liquor remaining after the ex traction of the fat, dissolving urobilinogen from the product so made by means of'a mixture of alcohol and chloroform that is a solvent for uroly or chloroform, evaporating the said solutionto dryness, this giving a residue high in percentage of urobilinogen, and adding benzoic acid to the said residue.

7. The method described in claim 6, the urobilinogen residue and the added benzoic acid be- It will be understood also that it is intended to cover all changes and modifications oi the example of the invention herein chosen for the purpose of illustrationwhich do not constitute departures from the spirit and scope of the invention. V

What I claim is: g 1. An antipressor serving when antipressor being substantially fat free and the proportion of the urobiiinogen and benzoic acid,

injected into. the human system to lower the blood pressure in ing made into the form of a hot aqueous solution, and the solution so made being filtered and then cooled, to cause precipitation of the antipressor composition. g

8. An antipressor composition, serving when injected into the human system to lower the blood pressure in cases of hypertension and pregnancy complicated by hypertension with symptoms of eclampsia, comprising a compound of benzoic acid with urobilinogen, the said composition beins substantially iat tree. 9. An antipressor serving when injected into the human system to lowerthe blood pressure in cases of hypertension and comprising the compounds remaining after the interaction of benzoic acid with urobilinogen, the antipressor being substantially fat free and the benzoic acid and urobilinogen being used in the proportion respectively, in the antipressor being not less than those required to show a'positive Ehrlichs test of approximately 0.5 to 2.5 parts of urobilinog'en to parts 01' benzoic'acid.

' HAROLD M. RABINOWITZ. 

